The research program is focused on the detailed mechanisms underlying the initiation of innate immune responses by the receptors and the ensuing signal transduction pathways. [unreadable] [unreadable] The innate immune system is the first line of defense against infection. It also initiates and directs the proper function of the adaptive immune response, the other branch of the immune system. The potency of the innate immune system has been harnessed as vaccination adjuvants against infections, cancer, and autoimmune diseases. Nonetheless, the underlying mechanisms of action only started to be delineated about ten years ago, when a major family of the innate immune receptors (the TLRs) was identified. It is now appreciated that both TLR-dependent and TLR-independent innate immune activation regulate adaptive immune responses. Among exciting progress in the innate immune filed, the recent crystal structures of the TLR2, TLR3 and TLR4 extracellular domains have provided a framework upon which further investigation of the innate immune recognition can be conducted. It is however apparent from these structural studies that the mechanisms of ligand recognition varies significantly among different TLRs. [unreadable] [unreadable] Our program integrates biochemical studies with extensive structural analysis of membrane bound (such as the Toll-like receptors) and cytoplasmic (such as RIG-I, NALP3 and ZBP1) receptors, either in complex with their ligands or downstream adapters/effector molecules. A critical feature of these innate immune receptors is that they distinguish among various classes of pathogenic molecules while retaining their capacity for responsiveness to a large number of related structures within a given biochemical class. How the binding domains of the innate receptors achieve such broad reactivity at the atomic level is one of the key issues this project addresses. Such information could be used to guide the development of new therapeutics that can either enhance or limit immune activation involving these receptors. Ligand binding by these receptors in turn initiates intracellular signaling cascades that ultimately lead to innate cellular responses that help fight infection and guide the adaptive immune responses. The project aims to decipher this signaling network through studying protein-protein interactions, using X-ray crystallography in conjunction with other biophysical and biochemical techniques. The ultimate goal is to not only delineate the mechanisms of innate immune responses at atomic details and contribute to our general understanding of signal transduction, but also to lay a foundation for future clinical exploitation of the innate immune system for human benefit, such as the development of more effective vaccine adjuvants.[unreadable] [unreadable] Progress has been achieved in the following areas during the year:[unreadable] [unreadable] 1). By taking advantage of a mammalian expression system, we have successfully expressed human TLR9 extracellular domain with HEK293 cells, in both adherent and suspension cultures. The suspension culture will allow for large-scale expression and purification for structural studies. In collaboration with Eicke Latz's lab at the University of Massachusetts, we achieved higher expression yield using a pre-protrypsin leader sequence for protein secretion. We are in the process of improving the expression yield further by utilizing a new vector from the Biotechnology Research Institute of the National Research Council, Canada. This vector was reported to increase protein expression several fold over our current expression vectors. The establishment of this high-yield suspension culture mammalian expression system will facilitate testing various expression constructs efficiently, for both membrane receptors and soluble/cytoplasmic receptors. [unreadable] [unreadable] 2). In collaboration with Jenny Ting from the University of North Carolina, we have successfully expressed and purified an amino-terminal fragment of NLR family member NALP12/Monarch-1. NALP12 attenuates the inflammatory responses by suppressing the activation of NF-kappaB upon TLR stimulation and serves as a crucial link between the TLR and NLR signaling pathways. We have achieved high level expression of NALP12 in both insect cells and mammalian cells (HEK 293). Crystallization of the NALP12 fragment has proven to be difficult as the sample becomes aggregation and precipitates at high concentration. We are currently testing complex formation with its downstream adapter ASC, as well as modifying the boundaries of the constructs. [unreadable] [unreadable] 3). By engineering bacteria expression vectors with multiple expression and purification tags, as well as utilizing metabolic regulation of gene expression from T7 promoters, we have achieved soluble expression of the TIRs from MyD88, TIRAP, TLR1, and TLR2, as well as a bacteria virulence factor TcpC that was reported to bind MyD88 through their TIR domains and thus compromise the innate immune response against bacterial infection. We have successfully crystallized the TIR domain of MyD88 and TcpC, and determined the structure of MyD88 TIR domain. We are in the process of determining the structure of TcpC TIR domain. The structure of the MyD88 TIR domain shows dramatic conformational differences compared with all other known TIR domain structures, whereas the crystal structure is very similar to recently deposited NMR structures of MyD88 TIR domain. This suggests that our crystal structure provides the basis for analyzing MyD88 function in the cytoplasm. [unreadable] [unreadable] 4). We have developed insights into the dimerization behavior of the MyD88 TIR domain. In the MyD88 TIR domain crystal, there is an intriguing TIR-TIR domain dimer formation through three long TIR domain loops, named "BB", "DD", and "EE" loops. These loops have been suspected to be involved in TIR domain dimerization and signaling. A number of important mutations in human and mouse TIR domain proteins map to these "BB"-"DD"-"EE" interface. We are verifying these interactions with NMR spectroscopy and yeast two-hybrid assay. [unreadable] [unreadable] 5). Using co-expression and co-purification strategies, we are analyzing the interactions of RIG-I family of receptors binding to their central adapter IPS-1. The goal is to obtain stable binding partners for structural and biophysical investigations. We are testing potential interacting pairs of different fragments of RIG-I, MDA5, LGP2, IPS-1, as well as ATG5 and ATG12 that were implicated in regulating RIG-I signaling. These potential binding partners are being tested with a bacterial expression system using different purification tags for each partner. Our preliminary results suggest that the presence of binding partners facilitated the expression of constructs previously difficult to express alone. [unreadable] [unreadable] 6). Similar to the strategy we adopted for the RIG-I family of receptors, we are studying binding of NLR family members binding to their central adapter ASC. Our previous recombinant expression studies have shown degraded ASC without binding partners. Recently we are able to detect stable expression of ASC in the presence of its binding partners. We have also produced expression constructs for insect and mammalian expression systems, particularly for larger fragments of ASC, NALP1, NALP3 and IPAF. We anticipate that samples from these eukaryotic expression systems will have less tendency to precipitate or aggregate, partially due to the authentic eukaryotic folding machinery. [unreadable] [unreadable] 7). Through a collaboration with Stefan Rothenburg at NICHD, we have been able to express both human and mouse ZBP1/DAI with our bacteria expression system. Our initial crystallization trials have produced interesting crystallization leads, but we have been unable to improve on the leads, possibly due to improper folding of the samples. We are producing constructs for insect/mammalian expression systems.